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Volume 272, Number 45, Issue of November 7, 1997 pp. 28590-28595

Presence of Laminin alpha 5 Chain and Lack of Laminin alpha 1 Chain during Human Muscle Development and in Muscular Dystrophies*

(Received for publication, August 19, 1997, and in revised form, September 5, 1997)

Carl-Fredrik Tiger Dagger , Marie-France Champliaud §, Fatima Pedrosa-Domellof , Lars-Eric Thornell , Peter Ekblom Dagger and Donald Gullberg Dagger par

From the Dagger  Department of Animal Physiology, Uppsala University, BMC, Box 596, S-751 24 Uppsala, Sweden, § The Cutaneous Biology Research Center, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA 02142-1299, and the  Department of Anatomy, Umeå University, S-901 87 Umeå, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin alpha 1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin alpha 1 chain. Nevertheless, there seems to be no expression of laminin alpha 1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin alpha 1 chain as a recombinant protein and made antibodies specific for human laminin alpha 1 chain (anti-hLN-alpha 1G4/G5). We also made antibodies to the human laminin alpha 5 chain purified from placenta. In the present report we show that hLN-alpha 1G4/G5 antibodies react with a 400-kDa laminin alpha 1 chain and that 4C7 reacts with a 380-kDa laminin alpha 5 chain. Immunohistochemistry with the hLN-alpha 1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin alpha 5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin alpha 1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.

INTRODUCTION

Cellular interactions with the extracellular matrix have been implied to be important for several stages of muscle development (1-4). An intact linkage to the surrounding basement membrane has been demonstrated to be of importance also for muscle stability in the adult stage (5, 6). During regeneration events following muscle damage, the basement membrane acts as a scaffold for the generation of new muscle fibers (7, 8). It is thus important to understand the molecular composition of basement membranes in muscle. Laminin-2, with the chain composition alpha 2, beta 1, gamma 1, is present in the muscle lineage from early stages of development in the mouse (9, 10) and is apparently the major laminin isoform in adult muscle basement membranes (11). The finding that genetic defects affecting laminin alpha 2 can cause muscular dystrophy has highlighted the importance of laminin-2 for the structural integrity of muscle (12, 13). Molecular compensation in certain forms of muscular dystrophies by increased expression of laminin chains may decrease the severity of the diseases. Some evidence for this has been obtained in immunohistochemistry studies with the antibody 4C7, which is one monoclonal antibody from a panel of antibodies raised against human placental laminins (14, 15). These antibodies were generated prior to the current knowledge about the existence of multiple laminin isoforms. The 4C7 antibody does not react with the laminin alpha 2 chain but has been considered to react with the human alpha 1 chain (14, 15). The antibody, commercially available under different names, has been widely used to detect human alpha 1 chain (previously called A chain) both in muscle and non-muscle tissues (14, 16-20). The 4C7 antigen has been detected in basement membranes of normal muscle, and in blood vessels in muscle tissue (21), and increased expression of it in muscular dystrophies has been documented in numerous reports (22-24). It thus seemed reasonable to suggest molecular compensation by alpha 1 chain in muscular dystrophies (22-24), particularly since many reports convincingly have shown that laminin-1 (alpha 1, beta 1, gamma 1) can stimulate proliferation, motility, and development of muscle cells in vitro (1, 25, 26). Nevertheless, we and others have failed to detect laminin alpha 1 chain in developing mouse muscle tissue (9, 27, 28), and no increased expression of this chain was seen in dystrophic mouse muscle (28). Furthermore, comparing the staining pattern in non-muscle tissue of 4C7 in human, rat, and hamster with the pattern seen in mouse and rat with other antibodies, there is a discrepancy. In non-neural tissues of mouse and rat the alpha 1 chain is largely confined to epithelial basement membranes (27, 29, 30), but the 4C7 antigen is widely distributed in developing and adult human, rat, and hamster tissues (15, 19, 20). The staining pattern of 4C7 in human tissues is also in disagreement with the distribution of the alpha 1 mRNA in human tissues (31, 32). Antibody 4C7 might thus detect some other alpha  laminin chain, but this proposal is speculative (33, 34) and has not been rigorously tested. Currently, five different laminin alpha  chains have been described (35) and 4C7 could potentially detect any of these or might detect several alpha  chains. In a cell line, 4C7 immunoprecipitated a large chain in the 400-kDa range together with 200-kDa chains, but the nature of the 400-kDa chain was not studied (15).

To clarify the discrepancies in laminin alpha 1 distribution in mouse and human tissues, we made antibodies to the recombinant E3 domain of human laminin alpha 1 and compared the specificity of these antibodies with that of 4C7. Immunoprecipitation of laminins from cell lines producing varying amounts of either alpha 1 or alpha 5 mRNA allowed a precise distinction of antibody specificity. Furthermore, we compared the distribution of laminin alpha 1 chain and the 4C7 antigen in developing human muscle and in dystrophic human muscle tissue. Since the distribution of the laminin alpha 1 chain in mouse kidney has been well described, we also analyzed the expression patterns in human kidney.

MATERIALS AND METHODS

Recombinant Laminin Expression

A 1180-base pair-long fragment from the 3'-end of human laminin alpha 1 chain (nucleotide residues 8140-9320) corresponding to the E3 region (carboxyl-terminal globular domain G4-G5) was amplified by PCR1 from a 4.5-kb laminin alpha 1 cDNA sequence ((36), clone number 7 supplied by E. Engvall The Burnham Institute, La Jolla Cancer Research Center) using AmpliTaq® (Perkin-Elmer). The primers were modified to include restriction sites for NotI and NheI to facilitate cloning into the expression vector. Primer sequences were as follows: forward primer, 5' GCC CCG CTA GCT CCC GAT GCA GAG GAC AGC A 3'; reverse primer, TCA GTT GCG GCC GCT CAG GAC TCG GTC CCA GG. The obtained PCR product was ligated into a TA-vector (PCR IITM, Invitrogen) for sequence confirmation. Sequencing was performed with a Pharmacia T7 SequencingTM kit (Pharmacia Biotech Inc.). The sequenced PCR product was cleaved with NotI/NheI and inserted into the episomal pCEP-Pu vector (which is a modified pCEP4 (Invitrogen) vector, provided by E. Pöschl Institute of Experimental Medicine, Friedrich-Alexander-University, Erlangen, Germany). The insertions sites were sequenced prior to transfection. 106 human embryonic kidney cells 293 EBNA (Invitrogen, Catalog number R-620-07) were stably transfected with 15 µg of hLN-alpha 1G4/G5 in pCEP-Pu using lipofectAMINETM reagent (Life Technologies, Inc.), according the instructions from the manufacturer. Transfected cells were selected in 2 µg/ml puromycin and 0.25 mg/ml G418 (Life Technologies, Inc.) and the medium from cells grown under serum-free conditions was analyzed for recombinant protein by SDS-PAGE. Purified protein was separated on a 10% SDS-PAGE under reducing conditions, visualized with Coomassie Brilliant Blue, excised, and digested "in-gel" with trypsin according to Ref. 37. Liberated peptides were further analyzed as described in Ref. 38. One peptide (SPQVQSFDFS) was analyzed and found to be identical with amino acids 3048-3057 in Ref. 36.

Antibodies

For the generation of antibodies to human laminin alpha 1, medium was collected from confluent 293 EBNA hLN-alpha 1G4/G5 cells under serum-free conditions and supplemented with 1 mM benzamidine, 1 mM EDTA, 1 mM N-ethylmaleimide. Collected medium was diluted 1:2 in water, passed over a 10-ml DEAE-Sepharose® Fast Flow (Pharmacia Biotech Inc.) column, serially connected to a 5-ml Hi trap Heparin-Sepharose® column (Pharmacia Biotech Inc.). The DEAE column was disconnected, and the heparin column was washed in 0.1 M NaCl, 20 mM Tris-HCl, pH 8.0, prior to eluting in 0.3 M NaCl in 20 mM Tris-HCl, pH 8.0. Peak fractions containing recombinant protein were concentrated on a second 1-ml Hi trap Heparin-Sepharose column, and the resulting peak fraction was used for immunizations of two rabbits, using 50-µg injections intramuscularly at intervals of 3 weeks. For immunohistochemistry, the antibodies were affinity-purified on the recombinant protein as described in Ref. 39 prior to staining. The polyclonal antibody to human laminin alpha 5 was generated as follows: an extract from human placenta was purified by affinity chromatography on a laminin beta 1 chain antibody (Ab 545) as described in Ref. 40. A major 380-kDa purified protein band on SDS-PAGE was cleaved with trypsin and microsequencing of resulting peptides revealed laminin alpha 5 sequences (see "Results"). The original 380-kDa SDS-PAGE band was used to generate the rabbit polyclonal antibodies to laminin alpha 5 chain. The monoclonal antibody recognizing laminin beta 1/gamma 1 chains (clone 4C12.8) was obtained from Immunotech. The polyclonal antibody to intact mouse laminin-1 was from Sigma (L9393). The 4C7 antibody (sold under the name mAb 1924) was from Chemicon. To visualize the proximal tubules a polyclonal antibody specific for a brush border antigen of proximal tubules was used (41).

Immunoprecipitation and Western Blotting

JAR cells (human choriocarcinoma cells ATCC No HTB-144), RD (rhabdomyosarcoma ATCC No CCL-136), WWCS-1 (Wilm's tumor cell line (42)), and G6 (cloned primary human fetal myoblasts (43)) were grown in Dulbecco's modified Eagle's medium under standard conditions. Cells were labeled overnight in in the presence of 25 µCi/ml [35S]methionine/cysteine (pro-Mix 35S cell labeling mix (Amersham Corp.)). Medium was collected from cells, centrifuged, and supplemented with protease inhibitors (1 mM benzamidine, 1 mM EDTA, 1 mM N-ethylmaleimide). The centrifuged medium was processed for immunoprecipitation as described (44). For Western blotting, conditioned medium was collected from JAR cells. Medium was passed over a Ricinus communis agglutinin I-agarose column (Vector Laboratories), washed extensively in phosphate-buffered saline, and eluted with 0.5 M D(+)-galactose (Sigma). Eluted proteins were directly used for immunoprecipitation. Immunoprecipitated proteins were solubilized in SDS-PAGE sample buffer and resolved on a 5% SDS-PAGE gel under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in a Trans-Blot cell (Bio-Rad). Membranes were incubated with primary antibody, washed in TBS + 0.05% Tween 20, followed by peroxidase-coupled sheep anti-rabbit IgG (Amersham) and developed using the ECL system (Amersham).

Immunohistochemistry

Serial sections, 5-8-µm thick, of muscle biopsies of boys referred for diagnostic purpose and shown to lack dystrophin, of muscle samples of human fetuses with a gestation age of 22 weeks, and of biopsies of human kidneys were cut in a Reichert Jung cryostat at -25 °C. The sections were collected on individual slides as well as on the same slide (one sample of human muscular dystrophy, one of human fetal muscle, and one of human kidney) to allow a direct comparison of staining intensity in the three different types of samples. The staining procedure was carried out as described in Ref. 28.

Northern Blot Analysis

Total RNA was isolated using Qiagen RNeasy midi kit according to the manufacturer's instructions. Northern blotting was performed as described (28). For laminin alpha 1, the 1180-base pair-long fragment used for recombinant laminin expression was used as a probe. For laminin alpha 5 a reverse transcription PCR amplified mouse cDNA (nucleotides 290-891) was obtained from newborn mouse kidney total RNA and used as a probe as described (30). In addition a 1.3-kb human EST clone (accession W67855) was obtained from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) consortium (I.M.A.G.E. consortium Clone ID:342926, United Kingdom Human Genome Mapping Project Resource Center, Hinxton, Cambridge, United Kingdom) and was sequenced from the vector T7 site and in a span of 360 nucleotides found to show 73% identity to mouse laminin alpha 5 chain (nucleotides 9874-10233) (45) and to be identical with a partial cDNA sequence for human laminin alpha 5 in nucleotides 1903-2262 (46). The opposite end of the EST clone was found to be identical to the untranslated end of the partial human laminin alpha 5 cDNA sequence (nucleotides 2891-3125) (46). The 1.3-kb fragment was excised with NotI/EcoRI and used as a probe.

RESULTS

Generation of Polyclonal Antibodies to Human Laminin-1 E3 Region

To obtain reagents specific for human laminin alpha 1 for immunohistochemistry on human tissues, we stably expressed cDNA coding for the E3 region (carboxyl-terminal globular domains G4-G5) of laminin alpha 1 episomally in 293 EBNA cells. Recombinant protein (hLN-alpha 1G4/G5) displayed an estimated molecular mass of 45 kDa under nonreducing conditions, shifted mobility to 55 kDa upon reduction, and bound heparin-Sepharose (Fig. 1). Following purification on DEAE- and heparin-Sepharose the hLN-alpha 1G4/G5 (Fig. 1) was used as an immunogen to generate polyclonal antibodies. These antibodies are further characterized below. We also generated an antibody to human laminin alpha 5 chain. The polyclonal rabbit antibody, anti-hLNalpha 5, was raised to a 380-kDa SDS-PAGE band purified from human placenta by immunoaffinity chromatography on anti-laminin beta 1 IgG (data not shown). Amino acid sequencing of tryptic peptides obtained from the 380-kDa band revealed two peptides, which were identified in the deduced amino acid sequence from a recently identified partial human laminin alpha 5 cDNA sequence (46) (Table I).

Fig. 1. Analysis of recombinant hLN-alpha 1G4/G5 protein by transfected 293 cells. Conditioned medium from [35S]methionine/cysteine-labeled nontransfected 293 EBNA cells (lanes a and b) and 293 EBNA-hLN-alpha 1G4/G5 cells (lanes c and d) was analyzed under nonreducing (lanes a and c) and reducing conditions (lanes b and d) on a 10% SDS-PAGE gel. Medium from [35S]cysteine/methionine-labeled cells was applied to heparin-Sepharose, and bound proteins from nontransfected (lane e) and transfected cells (lane f) were analyzed by SDS-PAGE. Coomassie Brilliant Blue staining of proteins in the medium fraction from 293 EBNA-hLN-alpha 1G4/G5 cells (lane g) and the DEAE/heparin-Sepharose-purified hLN-alpha 1G4/G5 protein (lane h). Position of molecular weight markers are shown.

[View Larger Version of this Image (73K GIF file)]

Table I. Amino acid sequences of tryptic peptides obtained from the 380-kDa band were identified as human laminin alpha 5, based on the deduced amino acid sequence from a partial human laminin alpha 5 cDNA (46)

Human laminin-alpha 5 partial KFYLQGPEPEPGQGTED
  cDNA residue #21  |||||||| ||| |||
Tryptic peptide  FYLQGPEPDPGQ-TED
Human laminin-alpha 5 partial     RQATGDYMGVSLR
  cDNA residue #46      ||||||||||||
Tryptic peptide      QATGDYMGVSLR

Northern Blot Analysis of Laminins

To characterize the antibodies we had generated we tested a number of cell lines in Northern blotting for their expression of laminin alpha 1 mRNA. As a comparison we also probed for laminin alpha 5 mRNA. Laminin alpha 1 mRNA was only detected in JAR cells, whereas laminin alpha 5 mRNA was detected at moderate levels in JAR cells and RD cells and and at lower levels in WCCS-1 cells (Fig. 2A). The size of laminin alpha 1 mRNA was estimated to approximately 10 kb, and the laminin alpha 5 mRNA, as detected by both laminin alpha 5 probes, was distinctly larger. The size was in agreement with the previously reported size of 11-12 kb in mouse (45).

Fig. 2. Laminin protein and mRNA analysis in different cell lines. A, aliquots of total RNA from JAR choriocarcinoma cells (J), RD rhabdomyosarcoma cells (R), and WCCS-1 Wilms tumor cells (W) were analyzed for laminin alpha 1 mRNA (hLNalpha 1) and laminin alpha 5 mRNA. Laminin alpha 5 mRNA was probed with a mouse probe (mLNalpha 5) and a human laminin alpha 5 probe (W67855). For control of equal loading, all lanes were hybridized with a probe to glyceraldehyde-3-phosphate dehydrogenase (G3PDH). B, JAR cells, RD cells, and WCCS-1 cells were metabolically labeled with [35S]cysteine/methionine and laminins were immunoprecipitated with antibodies to laminin beta /gamma chains (beta /gamma ), with antibodies to hLN-alpha 1G4/G5 (alpha 1), with polyclonal antibodies to human laminin alpha 5 chain (alpha 5), and with 4C7. Precipitated proteins were resolved on 5% SDS-PAGE gels under reducing conditions. The precipitates from JAR cells were analyzed on the same SDS-PAGE gel and RD and WCCS-1 on a separate gel. Molecular mass values in kilodaltons are indicated. The arrow denotes nonspecific band. C, conditioned medium from JAR cells was passed over RCA-I lectin-agarose, and bound proteins were immunoprecipitated with an antibody to laminin beta /gamma chains (lanes a and b) and with the 4C7 antibody (lanes c and d). Immunoprecipitated proteins were separated on a 5% SDS-PAGE gel under reducing conditions and blotted with the antibody to human LN alpha 1 (lanes a and c) or with a polyclonal antibody to laminin alpha 5 (lanes b and d). On a separate gel, immunoprecipitated laminins were silver-stained (lanes e-g). Proteins immunoprecipitated by anti-hLN-alpha 1G4/G5 (lane e), control with no primary antibody (lane f), and proteins precipitated with 4C7 (lane g) are shown. Nonspecific bands are denoted with arrows.

[View Larger Version of this Image (77K GIF file)]

4C7 Antibody Detects Laminin alpha 5 Chain

When laminins were immunoprecipitated with an antibody to laminin beta /gamma chains, two alpha  chain bands with molecular masses of 400 and 380 kDa were observed on SDS-PAGE from JAR cells (Fig. 2B). In contrast, the hLN-alpha 1G4/G5 antibodies only precipitated a complex containing 400- and 200-220-kDa bands.

The monoclonal antibody 4C7 has been suggested to recognize laminin alpha 1 chain. However, when medium from metabolically labeled JAR cells was immunoprecipitated with anti-hLN-alpha 1G4/G5 and 4C7 in parallel, different laminin alpha  chain bands were obtained. Whereas anti-hLN-alpha 1G4/G5 precipitated the 400-kDa band, 4C7 precipitated a 380-kDa band (Fig. 2B). In mouse the laminin alpha 5 chain has been reported to have an molecular mass of 380 kDa (47). From JAR cells anti-hLNalpha 5 precipitated a 380-kDa band (Fig. 2B). From RD cells lacking reactivity with anti-hLN-alpha 1G4/G5, antibodies to laminin beta /gamma chains and anti-hLNalpha 5 still precipitated a laminin with a molecular mass of the alpha  chain of 380 kDa. A 380 kDa alpha  chain band was also precipitated from RD and G6 cells with 4C7 (data not shown). WWCS-1 cells, shown in Northern to lack laminin alpha 1 mRNA and to express low levels of laminin alpha 5 mRNA, only precipitated visible beta /gamma chain complexes in immunoprecipitation under the conditions used.

Western blotting of JAR cell medium and proteins immunoprecipitated from this medium with antibodies to laminin beta /gamma chains, and subsequently blotted with antibodies to hLN-alpha 1G4/G5 (Fig. 2C, lane a), revealed strong reactivity with the 400-kDa band. Anti-hLNalpha 5 reacted weakly with a 380-kDa band in the material precipitated by laminin beta /gamma chains (lane b). As shown in C the material immunoprecipitated from JAR cells with the 4C7 antibody did not react with hLN-alpha 1G4/G5 antibodies (lane c), whereas Western blotting with anti-hLNalpha 5 resulted in reactivity with the 380-kDa band (lane d). We also performed silver staining to independently illustrate the size difference between the laminin alpha  chains precipitated by the two antibodies. Silver staining of proteins immunoprecipitated with anti-hLN-alpha 1G4/G5 revealed a distinct 400-kDa band together with 200-220-kDa bands (lane e), whereas silver staining of 4C7 reactive material revealed the 380-kDa band in addition to 200-220-kDa bands (lane g). The 200-220-kDa bands were recognized in Western blotting by a polyclonal antibody to mouse laminin alpha 1beta 1gamma 1 chains (data not shown).

Distribution of Laminin alpha 1 and Laminin alpha 5 in Fetal and Adult Human Tissues

When human adult kidney was stained with affinity-purified antibodies to human laminin alpha 1 (anti-hLN-alpha 1G4/G5) and human laminin alpha 5 (4C7), contrasting staining patterns were observed. Anti-hLN-alpha 1G4/G5 selectively stained a subset of proximal tubuli, whereas 4C7 stained proximal and distal tubuli, glomerular basement membranes, and blood vessels (Fig. 3, A-C). In agreement with previously reported data, 4C7 stained muscle fibers in addition to blood vessels in human fetal muscle (Fig. 4A). In biopsy material from a dystrophic DMD boy, 4C7 stained basement membranes of muscle fibers, blood vessels, and somewhat more intensely groups of small diameter regenerating muscle fibers (Fig. 4B). In contrast, the hLN-alpha 1G4/G5 antibody did not specifically stain either developing or dystrophic muscle tissue (Fig. 4, C and D).

Fig. 3. Immunohistochemical analysis of laminin alpha 1 and laminin alpha 5 in human kidney. Immunofluorescence with 4C7 on human kidney sections revealed human laminin alpha 5 in tubuli, glomeruli, and blood vessels (A). Double immunofluorescence revealed that anti-hLN-alpha 1G4/G5 only stained a subset of tubuli (B). Staining of a paralell section with a proximal tubuli brush border marker (C) revealed that the laminin alpha 1-positive tubuli were proximal tubuli. Glomerulus is depicted with an arrow. Bar: 200 µm.

[View Larger Version of this Image (75K GIF file)]

Fig. 4. Detection of laminin alpha 1 and laminin alpha 5 in human skeletal muscle. Immunofluorescence with 4C7 on cross-sections of human fetal muscle revealed the presence of laminin alpha 5 in muscle fiber basement membranes and blood vessels (A), whereas a parallel section lacked detectable levels of human laminin alpha 1 (C). In DMD patient sections 4C7 stained larger blood vessels, capillaries, and around small myofibers (B), whereas laminin alpha 1 was not detected in a parallel section (D). Bar: 100 µm.

[View Larger Version of this Image (93K GIF file)]

DISCUSSION

In some forms of muscular dystrophy the primary defect is a disturbed linkage between the muscle fiber and the basement membrane (5, 6). This leads to muscle degeneration but also to a regeneration event where satellite cells are activated, replicate, and fuse to form new myofibers. During this process the basement membrane is used for migration and as a scaffold for the formation of new fibers (7, 8). Little is known about the basement membrane components made during regeneration. Laminins (35) and collagen IV (48) exist as multiple genetically distinct isoforms, and agrin is subject to extensive alternative splicing (49), opening up a wide spectrum of possible structural variations in the basement membranes synthesized by regenerating muscle fibers. One possibility is that some forms of laminins are up-regulated during regeneration. In a study of DMD, it was found that the laminin recognized by the antibody 4C7 was expressed in regenerating areas (23). The 4C7 antigen was also found to be induced in laminin alpha 2-deficient congenital muscular dystrophy (24). Since 4C7 has been reported to detect laminin alpha 1 chain, the results have been interpreted as an up-regulation of alpha 1 chain in these muscular dystrophies. Nevertheless, no clear up-regulation of laminin alpha 1 chain was seen in a mouse model of muscular dystrophy (28). The discrepancy seems to be due to antibody specificity, since we demonstrate here that 4C7 detects human laminin alpha 5 chain and not the alpha 1 chain. This gradually became apparent during our efforts to study laminin alpha 1 chain expression in human muscular dystrophies.

To study a possible up-regulation of laminin alpha 1 chain in human muscle diseases, we raised an antibody against a recombinant fragment of human laminin alpha 1 chain. A cDNA clone covering the most carboxyl-terminal globular domains (G4-G5) of the alpha 1 chain was transfected into mammalian cells to produce recombinant protein hLN-alpha 1G4/G5. This was used as an immunogen to produce polyclonal antibodies. The antibodies against hLN-alpha 1G4/G5 did not react with human muscle tissues. Using several assays, we therefore tested whether 4C7 shows a reactivity similar to the hLN-alpha 1G4/G5 antibody. We made a polyclonal antibody against purified human laminin alpha 5 chain for comparison. Medium from cell lines, which by Northern blotting could be shown to produce variable amounts of either alpha 1 or alpha 5 mRNA, were used for immunoprecipitation with the different antibodies. JAR cells, which produced both chains, proved particularly important for this analysis, and other cell lines served as excellent controls. From the medium of JAR cells, our hLN-alpha 1G4/G5 antibody and 4C7 antibody precipitated heterotrimers with different alpha  chains. Both antibodies precipitated two similar 200-kDa bands assumed to be beta 1 and gamma 1 chain, based on reactivity in Western blotting with antibodies to mouse laminin-1 (recognizing human beta 1/gamma 1 chains in immunoblotting). Whereas precipitates obtained with antibody hLN-alpha 1G4/G5 contained a polypeptide with the expected molecular mass of 400 kDa, those of 4C7 contained a slightly smaller 380-kDa polypeptide. Recent studies in mouse have shown that the alpha 5 chain is slightly smaller than alpha 1 (47). Silver staining of immunoprecipitated material on gels also revealed that hLN-alpha 1G4/G5 antibody detected a larger protein than 4C7. It has been suggested recently that 4C7 might recognize the alpha 1 chain together with other chains (45), but we found no evidence for reactivity of 4C7 with alpha 1 chain in either assay. By immunoblotting, the 380-kDa polypeptide could be identified as laminin alpha 5 chain. The used antibody to laminin alpha 5 was raised against an excised 380-kDa SDS-PAGE band, which by peptide microsequencing was identified as human laminin alpha 5. Moreover, in cell lines shown by Northern blotting to produce low amounts of alpha 5 chain mRNA, we failed to immunoprecipitate any bands with 4C7 or the polyclonal antibody to laminin alpha 5. Finally, we tested the different antibodies in immunofluorescence and found that antibody hLN-alpha 1G4/G5 and 4C7 gave the same expression pattern in human tissues as antibodies against mouse laminin alpha 1 chain and alpha 5 chain in mouse tissues. These results are in agreement with the mapping of all five laminin alpha  chains by Miner et al. (34), which concluded that alpha 5 was the most widely expressed, and alpha 1 was the most restricted. Based on these studies we conclude that our hLN-alpha 1G4/G5 antibody detects human alpha 1 chain and may at present be the only antibody with a documented true specificity toward human alpha 1 chain, which is useful in immunohistochemistry, Western blotting, and immunoprecipitations. In contrast, we show that 4C7 is a specific monoclonal antibody for laminin alpha 5 chain. It is likely that the other monoclonal antibodies described by Engvall et al. (15) to detect larger 300-400-kDa laminin chains also detect alpha 5 chain, but this remains to be shown. The identification of laminin alpha 5 chain as the sole antigen of 4C7 antibody clarifies several much debated issues concerning the distribution of laminin alpha  chains.

The implication of our current findings for previous data obtained in muscle is thus that human laminin alpha 5 is expressed in developing muscle and that this "embryonic" muscle laminin isoform is re-expressed in the regenerating muscle basement membrane in dystrophic muscle. In contrast, laminin alpha 1 cannot be detected in human fetal or regenerating muscle basement membranes, in accordance with previous data from mouse tissues (9, 27, 28). In dystrophin-deficient forms of muscular dystrophy, a recent therapeutic approach is to try and induce expression of the dystrophin homologue utrophin (50). A similar approach might be feasible in laminin-2-deficient congenital dystrophies, where a compensatory up-regulation of another laminin at an early stage of the disease might be beneficiary. Laminin-1 (chain composition alpha 1, beta 1, gamma 1) clearly stimulates myogenesis in vitro (1, 25, 26). In the light of the present findings it might nevertheless be more relevant to test whether laminin-10 (chain composition alpha 5beta 1gamma 1) can stimulate myogenesis in vitro. Moreover, it will be essential to determine whether the induction of laminin alpha 5 reduces the severity of the disease in laminin-2-deficient congenital muscular dystrophy.

The current findings have broad implications also for several open issues concerning the nature of laminin isoforms in non-muscle tissues. One of the few major controversies in the laminin field has been the seemingly simple issue of the distribution of the alpha 1 chain. This seems to be largely resolved by the current results. The 4C7 antibody, commercially available under different names, has been much used to study the distribution of laminin alpha 1 chain in human, hamster, rat, and guinea pig tissues. The 4C7 antibody described by Engvall et al. (15) should not be confused with a more recent, well described laminin antibody recognizing laminin-5 (alpha 3beta 3gamma 2) also named 4C7 (51). A data base search revealed numerous publications that have used the first described 4C7 antibody to demonstrate a broad tissue distribution of the antigen. All these results are different to the findings of a somewhat more limited distribution of alpha 1 chain in mouse and rat embryonic and adult tissues (27, 29, 30, 52). Here we therefore studied the distribution of laminin alpha 1 chain in adult human kidney with our antibody against recombinant human E3 fragment. It selectively stained the basement membranes of a subset of the proximal tubules. It is highly significant that no staining was seen in basement membranes of blood vessels, distal tubules, or the collecting ducts. This is in complete agreement with our previous findings in mouse (29) and rat kidney (30). These studies strongly suggest that the distribution of laminin alpha 1 chain is similar in rodent and human adult kidneys. Based on our studies in muscle and kidney, we predict that the staining pattern in other tissues with anti-hLN-alpha 1G4/G5 or with other true alpha 1 chain-specific antibodies will match the pattern of alpha 1 immunoreactivity in mouse. This will be of some importance to study in the future, given the reports of the distribution of laminin alpha 1 chain performed with antibody 4C7. Many of the studies performed with 4C7 antibody should be seen as valuable sources for descriptions of the distribution of alpha 5 chain. It should be noted, however, that in the mouse there are two alpha 5 mRNA transcripts, 9 and 12 kb, indicating the existence of two laminin alpha 5 isoforms (34). Whether such isoforms exist in human is unclear, since we have so far noticed only one 12-kb mRNA in the cells we studied. We do not know whether 4C7 detects different splice variants of the alpha 5 chains and whether they exist in human tissues. Although this particular detail is still unclear, it is evident that 4C7, that reacts at least with human, horse (own data), guinea pig, rat, and hamster tissues, will be useful for many studies of the major alpha 5 chain isoform in many species.

FOOTNOTES

*   This work was supported by Grants 12x-10817 (to D. G) and 12x-3934 (to L.-E. T.) from The Swedish Medical Research Council and the Konung Gustav V:s 80:års fond (to D. G)., Knut and Alice Wallenberg Foundation (to P. E.), and Cancerfonden (to P. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed. Tel.: 46-18-4714175; Fax: 46-18-508095; E-mail: donald.gullberg{at}zoofys.uu.se.
1   The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; DMD, Duchenne muscular dystrophy.

ACKNOWLEDGEMENTS

The skillful technical assistance of P. Jalonen is acknowledged. We also thank E. Pöschl for the expression vector, E. Engvall for human laminin alpha 1 cDNA, and E. Larsson for providing the human kidney tissue. The technical expertise of B. Ek, for the amino acid sequence analysis, is acknowledged.

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